Identification of the most efficient protocol according to gene expression. (a) Heatmap representation of 20 neuron- and/or dopamine-specific genes (KCNJ6, GPHN, SYP, SLC6A3, SLC18A2, SCNCAIP, RBFOX3, PITX3, PLXNC1, DLG4, DDC, DRD2, LMX1B, EN1, NR4A2, LMX1A, CORIN, NES, GJB2, and CNST) for 10 different protocols, labeled A, A2, B, B2, C, CA2, CB2, D, E, and EA2. Green: underexpressed genes; red: overexpressed genes. Time point labels correspond to Day -15 = untreated stem cells; Day 0 = start of cell differentiation, after an induction period of at least 15 days for protocols A, A2, B, B2, C, CA2, and CB2; durations of differentiation periods are indicated (Day 2 to Day 46). Protocol B2, framed in white, induced the most important overexpression of dopaminergic (DA) neuron-specific transcripts. (b) Schematic representation of the six step-based protocol B2 showing the basic medium (normal font), small molecules (italics), and the coatings with fetal calf serum (FCS), until Day 2, and poly-L-ornithine/fibronectin/laminin from Day 2.